Drug Delivery Using Liposomes Over Conventional Delivery Methods
Drug delivery methods have become a hot topic of focus due to the difficulty of targeting medical interventions with small or large molecules alone. As small molecule drugs are designed and tested, an incredible number of possible candidates are removed from consideration due to issues involving poor pharmacodynamics or kinetics (1). A similar issue exists in larger molecular drugs based on DNA, RNA and proteins. Studies involving the direct injection of plasmid DNA and smaller strands of RNA have shown that nucleases in blood serum destroy these drugs in a highly efficient manner (2). As such, nucleic acids cannot be delivered into the body without substantial help. Nucleic acid analogues have been used to overcome blood instability issues by being resistant to nucleases (3). However, the nucleic acid analogue approach still suffers from issues involving delivery into cells. When foreign protein drugs are delivered into the body, they are usually targeted by the humoral immune system (4). For example, patients with haemophilia A and B receiving injections of blood coagulation factors frequently develop neutralizing antibody responses which in many cases render the treatments useless with time (5). Although incredible genetic discoveries in various medical fields have been made over the last decade and a half, the lack of a practical delivery method for large molecules renders this data largely unusable. As a result, years of work have been put into designing methods to solve the issue of large molecule delivery. These methods mostly include the use of modified recombinant viruses and non-viral liposomes (6). Although viruses have shown promise under some medical circumstances there are ongoing issues of toxicity, target specificity or cell tropism, and ethical concerns associated with their use (7). Virus particles also become targets for the humoral immune system, greatly limiting their use to a narrow time window before neutralizing antibodies arise (8). If treatments are unsuccessful in this time window, further virus injections are quickly eliminated by the immune system. Non-viral methods based on liposomes have been seen as the safer alternative to viral delivery of large molecules. Efforts involving liposomes have been classically hampered by low efficiency of delivery, issues with vehicle loading efficiency, and practical issues involving preservation of structure during storage (3, 9).
Liposome Design Challenges with Delivery Efficiency, Drug Loading, and Structure Preservation
Liposomes are normally single phospholipid bilayer bubbles which separate the drug inside from the hostile environment outside. Incredible arrays of methods have been used to modify the outside of liposomes in order to make them more targetable, more delivery efficiency, and more structurally stable. Efforts to target the liposome via linked antibodies/peptides to the surface of the desired cell type for endosome uptake are known (10). When the targeted cell envelopes the liposome, the newly formed endosome continues through a natural process which degrades the contents for use as food sources. During this process, the contents of the liposome are freed into the cytoplasm usually via the destruction of the endosome membrane. The release of late-endosomal or lysosomal contents into the cytoplasm is a source of cytotoxic effects of non-viral vectors (11). Now in the cytoplasm, the liposome contents are then free to act in the local environment. However, targeting the nucleus in non-mitotic cells is impeded by the dual-bilayered nuclear membrane barrier and the nuclear pore complex. Some have attempted to address this issue by introducing transcription factor binding sites into the therapeutic DNA drug (12). Transcription factors bind to target sites on the DNA drug and help to shuttle it to the nucleus during signal transduction. These efforts have increased the rate of uptake into the nucleus; however the efficiencies of liposome therapy still remain much lower than robust viral methods. The movement of nucleic acids into the nucleus from the cytoplasm is dependent on the length of the strand due to the need for this molecule to fit though the narrow nuclear pore complex.
Another additive to the liposome is the use of membrane fusion proteins. These proteins have been employed to allow liposomal membranes to fuse with the cell's plasma membrane. This method unloads the contents of the cell into the cytoplasm without breaking down endosomal barriers, avoiding considerable cytotoxicity (13, 14). In effect, the liposome can be loaded with any sized nucleic acid for delivery into the cytoplasm. However, nucleic acids delivered by membrane fusion proteins into the cytoplasm are still subject to the “thread in needle” efficiency of the nuclear pore complex.
The efficiency of drug loading and structure retention can also be of concern in the practical implementation of liposomal drug delivery. In simplest terms, a tiny bubble can be formed easily, but keeping it intact during drug loading and long term storage has provided challenges (9). When a normal liposome mixture is extruded to produce the desired single membrane unilamellar format, the subsequent drug loading and storage can become a problem. Freeze dried liposomes do not retain their original shape and size upon addition of soluble drug. Since their shape and size are relevant to the desired delivery function, liposomes should be shape-stabilized prior to storage (9). In order to address this issue, some have used complexing agents to harden the liposome membrane/structure. This allows liposomes to be formed with one membrane, to be fixed in that structure through a complexing agent, freeze-dried, and then loaded by hydrodynamic force (drug in water) (15). This allows a liposomal drug to be assembled and stored as a freeze dried mixture and loaded efficiently prior to use. Although, the efficiency of the liposome assembly process is enhanced, surface hardening and complexing chemistry is incompatible with the use of membrane fusion protein methods. This is because the modified liposome surface has no fluidity or opening within which a membrane fusion protein could function. This lack of fluidity limits externally hardened liposomes to a delivery efficiency which is dependent on endosomal escape.
Self-Assembling Structures Based on DNA Complementary Binding
In the past decade, researchers have begun to explore the assembly of complex structures at the nanometer scale. Nanotechnology often employs information encoding DNA to design structures with preset parameters (16). Structures often are designed by linking ssDNA to effector molecule(s) and then assembled by the complementary base pairing innate to dsDNA. These techniques coupled with lithographic chip and microfluidics gives the user the capability to create devices that can perform a wide array of tasks from sensing DNA sequences, to the step wise assembly of small machines (17). The use of nanotechnology in the synthesis of drugs is still in its infancy. To date, only a few labs have considered the use of DNA in the synthesis of drug delivery vehicles. These interests surround designing DNA to form three dimensional structures which are then placed in the body (18). However, most of these techniques do not account for the body's innate ability to clear foreign DNA from the blood stream. Liposomes, which are used to hide DNA cargo from destructive blood enzymes, have not been designed with a self assembling platform.
There is a need in the art for a liposome and method of preparing a liposome with an internal structure that is amenable to the addition of surface proteins with a fluid membrane, and while still being stable during a freeze thaw cycle. In addition, there is a need in the art for an internal structure in a liposome that could aid its overall structural stability by keeping membrane layers from mixing and moving during both the freeze-drying and storage. Further, there is a need in the art for a liposome having an internal structure that could also aid in the synthesis of the overall vehicle with multiple bilayers and by allowing effector proteins to bind to the scaffold itself, and the product of which could be capable of taking any sized molecule and placing in any sub-cellular address in the body.